Peptide useful as a ligand

ABSTRACT

A peptide of formula (I) 
     
         (H.sub.2 N--X.sub.1 --Thr--X.sub.2 --CO).sub.n --R         (I) 
    
     where 
     X 1  and X 2 , different one another, are an amino acid residue of arginine or tyrosine in configuration L or D, wherein the hydroxy group of threonine and tyrosine and the guanidine moiety of arginine may be protected by a compound conventionally used in peptide chemistry for protecting the hydroxy group and the guanidine moiety, respectively, n is 1, 2, 3 or 4, and 
     R, when n is 2, 3 or 4, is a group suitable for forming a dimer, trimer or tetramer, while, when n is 1, R is OH, a single amino acid residue, or a peptide chain containing up to 7 amino acid residues.

This invention relates to a peptide useful as a ligand, the process forpreparing thereof, and the use thereof as a immunoglobulins ligand.

More particularly, the present invention relates to a peptide capable ofbinding non covalently itself to the constant portion ofimmunoglobulins.

Immunoglobulins, also known as antibodies, are extremely important indiagnostic and therapeutic field. Indeed, in the first case they arewidely used as reagents useful for the identification and quantificationof compounds in biological fluids, while in the second case they areused as agents capable of binding themselves to biological moleculesinvolved in physiological processes of therapeutic significance. In viewof the above mentioned significance, their production, and above alltheir purification, are extremely important from an industrial point ofview.

Immunoglobulins can be obtained from animal sera, or from cultivation ofsuitable cell lines.

Their purification is carried by means of conventional chromatographictechniques, such as ionic exchange or gel filtration, or preferably byaffinity chromatography using columns prepared by immobilization ofprotein A, obtained from Staphylococcus aureus, which is capable ofbinding specifically itself to the constant portion of immunoglobulinsSiodahl, J. Eur. J., Biochem 78: 471-490 (1977)!. However, protein Asuffers from many limitations when used on a large scale since itsextractive origin calls for a careful control and a careful purificationin order to avoid contamination of the product purified using saidprotein. In addition, protein A is not very stable to denaturingconditions and in the presence of agents used to remove biologicalcontaminants such as viruses or nucleic acid fragments. Finally, theproduction cost of protein A is extremely high and limits its use inpurifications on a large scale.

Therefore, there is still a great need for a synthetic ligand capable ofmimicking protein A as far as the ability to recognize the constantportion of immunoglobulins is concerned, which however can bemanufactured at low cost. Moreover, thanks to the synthetic origin, itwould be devoid of biological contaminants.

It has now been found that these properties are shown by a peptidecomprising the amino acid residues of arginine, threonine and tyrosine.

In particular, it has been found that the above mentioned properties areshown by a peptide comprising the sequence:

    --HN--X.sub.1 --Thr--X.sub.2 --CO--                        (S)

where

X₁ is an amino acid residue of arginine or tyrosine having configurationL or D,

X₂ is an amino acid residue of tyrosine or arginine having configurationL or D,

SUMMARY OF THE INVENTION

Thr is an amino acid residue of threonine having configuration L or D,provided, however, that X, is arginine when X₂ is tyrosine, and X, istyrosine when X₂ is arginine.

Preferably, at least one amino acid residue of the sequence (S) has Dconfiguration.

Even more preferably, two or all the three amino acid residues of thesequence (S) have D configuration.

It is therefore a first object of this invention to provide a peptide offormula (I)

    (H.sub.2 N--X.sub.1 --Thr--X.sub.2 --CO).sub.n --R         (I)

where

X₁ and X₂, different one another, are an amino acid residue of arginineor tyrosine in configuration L or D, wherein the hydroxy group ofthreonine and tyrosine and the guanidine moiety of arginine may beprotected by a compound conventionally used in peptide chemistry forprotecting the hydroxy group and the guanidine moiety, respectively,

n is 1, 2, 3 or 4, and

R, when n is 2, 3 or 4, is a group suitable for forming a dimer, trimeror tetramer, while, when n is 1, R is OH, a single amino acid residue,or a peptide chain comprising up to 7 amino acid residues.

As used herein the terms "dimer", "trimer" and "tetramer" are intendedto mean a peptide comprising 2, 3, or 4 sequences (S).

A typical example of a group suitable for forming a dimer (n=2) is alysine residue. A typical example of a group suitable for forming atrimer (n=3) is a dipeptide lysil-lysine of formula Lys-Lys. A typicalexample of a group suitable for forming a tetramer (n=4) is a branchedtripeptide of formula Lys-Lys(ε-Lys).

A typical example of a tetramer of formula (I) has the following formula

    (H.sub.2 N--X.sub.1 --Thr--X.sub.2 --CO).sub.4 --Lys.sub.2 --Lys--Gly--OH (IA)

where

X₁ and X₂ have the above mentioned meanings and wherein the hydroxygroup of threonine and tyrosine and the guanidine moiety of arginine maybe protected by a compound conventionally used in peptide chemistry forprotecting the hydroxy group and the guanidine moiety, respectively.

Many protecting groups for protecting the hydroxy group in peptidesynthesis are reported in the literature (G. A. Grant, Syntheticpeptides: a user's guide, Freeman, N.Y., 1992).

Typical examples of said protecting groups are: ter-butyl (tBu) (LaJoie, G. Crivici, A., Adamson, J. G. "Synthesis" 571-572 (1990)) and thebenzyl group (Yojima "Tetrahedron" 44:805-819 (1988)).

Many groups useful for protecting the guanidine moiety of arginine arealso known from the literature (Grant, G. A. Synthetic peptides: Auser's guide, Freeman, N.Y., 1992).

Typical examples of said protecting groups are:2,2,5,7,8-pentamethylcroman-6-sulphonyl (Pmc) and4-methoxy-2,3,6-trimethylbenzene (Mtr) (Ramage & Green, "TetrahedronLetters", 28,2287 (1987); Fujino et al. "Chem. Pharm. Bull.", 29,2825(1981).

Typical examples of thus protected compounds of formula (I) are thecompounds Boc--D--Arg(Pbf--D--Thr(tBu)--D--Tyr(tBu)--OMe of Example1(d), and (H₂ N--Arg(Pmc)--Thr(OtBu)--Tyr(OtBu)--CO)₄ --Lys₂--Lys--Gly--OH of Example 2.

When n is 1 and R is a peptide comprising from one to seven amino acidresidues, all the amino acids comprised in the sequence may be differentor equal to each other and have L or D configuration. The Dconfiguration is the preferred one. Furthermore, simple and cheap aminoacids will be preferred.

Specific examples of R for n equal to 1 are, Gly or Ala, Gly-Gly,Gly-Ala, Ala-Gly, Ala-Ala, Gly-Gly-Gly, Ala-Ala-Ala, Gly-Gly-Gly-Gly(SEQ ID NO:1), Gly-Gly-Gly-Gly-Gly (SEQ ID NO:2), Gly-Ala-Gly-Ala-Gly(SEQ ID NO:3), Ala-Gly-Ala-Gly-Ala-Gly-Ala (SEQ ID NO:4).

The peptides of formula (I) may be readily prepared according to boththe conventional liquid phase peptide preparation and solid-phasepeptide preparation techniques.

The preparation according to the solid-phase technique is preferablycarried out by means of an automatic synthesizer. A typical example of asuitable automatic synthesizer is the model 431 A from AppliedBiosystems (Foster City, Calif., USA). Preferably, the preparation isperformed according to the synthesis procedures recommended by themanufacturer, said procedures being usually based on known methods welldescribed in the literature (Atherton & Sheppard, 1989, Solid PhasePeptide Synthesis: A practical approach, IRL Press, Oxford).

It is a third object of this invention to provide the use of a compoundof formula (I) to form complexes with at least one immunoglobulin in aseparation process of said immunoglobulin or mixture of immunoglobulins.

Examples of immunoglobulins capable of forming complexes owing to noncovalent binding to compounds of formula (I), are: mouse IgG, rat IgG,chicken IgY, goat IgG, bovine IgG, human IgG, human IgA, and of otherspecies, human IgM and of other species.

A typical example of a method for the separation and purification of animmunoglobulin comprises:

(i) immobilizing on an affinity chromatography support a compoundcapable of binding non covalently itself to at least one immunoglobulin,

(ii) packing said affinity chromatography support in a chromatographiccolumn,

(iii) equilibrating said column with a buffer capable of promoting aninteraction between immunoglobulin and the immobilized compound,

(iv) loading said column with a fluid comprising at least oneimmunoglobulin,

(v) washing said column with at least one liquid capable of eluting theimpurities without interfering with the interaction betweenimmunoglobulin and the immobilized compound,

(vi) eluting said immunoglobulin previously adsorbed on the column witha dissociating eluent,

and is characterized in that:

the compound capable of binding itself non covalently to at least oneimmunoglobulin is a compound of formula (I), where X₁, X₂, n and R havethe meanings shown above.

Steps from (i) to (vi) are carried out according to conventionaltechniques.

Preferably, the support for affinity chromatography is preactivated withepoxide groups for direct coupling to peptides and proteins. Typicalexamples of suitable supports are the resin activated-CH SEPHAROSE™ 4Bfrom Pharmacia (Sweden), the resin PROTEIN-PAK™ (Waters, USA) the resinEUPERGIT™ C30 N (Rohm & Haas, Germany), or AFFI-GEL™ from BioRad (USA).

Step (i) is preferably carried out in the presence of a weakly basicbuffer solution having a pH value of from 8.5 to 9.0.

Step (iii) is preferably performed with a neutral buffer such as, forexample, a 25 mM Bis-Tris solution having pH 6.5, or a 50 mM phosphatebuffer solution having pH 7.0.

Step (v) is preferably carried out by using a neutral buffer having alow ionic strength such as, for example, a 25 mM Bis-Tris solutionhaving pH 6.5.

Examples of dissociating eluents useful in step (vi) comprise acid orbasic aqueous solutions. Typical examples comprise aqueous solutions ofacetic acid at pH 2.5 or of sodium bicarbonate at pH 9.0.

This separation and purification technique is widely described in theliterature Narayanan, S. R., "Preparative affinity chromatography ofproteins" J. Chromatogr., 658:237-258 (1994), as well as referencesquoted therein; Lowe, C. R., "Laboratory technique in Biochemistry andMolecular Biology", Work and Burdon, vol. 7, part 2, Elsevier, N.Holland, Amsterdam; Ey et al. Immunochemistry, 15:429 (1978)!.

The compounds of this invention may also be used in the qualitative orquantitative determination of immunoglobulins according to the wellknown ELISA technique.

A typical example of a method for quantitative determination of animmunoglobulin or a mixture of immunoglobulins according to the ELISAtechnique comprises:

(1) immobilizing a compound capable of binding itself non covalently toat least one immunoglobulin on a microtiter plate for ELISAdetermination,

(2) incubating a sample containing the immunoglobulin or theimmunoglobulins to be determined on said microtiter plate,

(3) washing said microtiter plate,

(4) detecting the thus formed immobilized complexcompound/immunoglobulin,

and is characterized in that

the compound capable of binding itself non covalently to at least oneimmunoglobulin is a compound of formula (I) where X₁, X₂, n and R havethe above mentioned meanings.

The analytical determination of immunoglobulins according to the ELISAtechnique is widely described in the literature ("Immunochemistry inpractice", Johnstone & Thorpe, (1987), Blackwell, Oxford, UK).

Preferably, step 1 is carried out using a plastic microtiter plate suchas, for example, of PVC, with 96 well filled with 0.1M sodiumbicarbonate solutions having pH 9.0 and containing variable amounts of aligand (0-50 μg/well). After 24 h incubation, excess solution isremoved, and the microtiter plates are washed with phosphate buffer andthe wells are filled with a 3% bovine albumin solution to eliminate aspecific interaction sites.

In step 2, microtiter plates are washed with phosphate buffer and thewells are filled with solutions containing an immunoglobulin, preferablyderivatized with biotin. Microtiter plates are then incubated for 4-18 hat 20°-37° C.

Washing in step 3 is preferably carried out with phosphate buffer.

Step 4 is performed by adding to each well a solution of avidinconjugated to peroxidase. After 2 h incubation, microtiter plates arewashed, preferably again with a phosphate buffer. Then a solution ofo-phenylenediamine is added and color formation is detected with asuitable ELISA reader.

These and further features of the present invention will result moreclearly from the following examples and the enclosed figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the purification of rabbit immunoglobulin from crude serumusing affinity chromatography on a column prepared by immobilization ofthe compound of formula (H-L-Arg-L-Thr-L-Tyr-)₄ -Lys₂ -Lys-Gly-OH;

FIG. 2 shows the electrophoretic analysis on polyacrylamide gels of thefractions deriving from the purification of rabbit immunoglobulins byusing the compound of formula (H-L-Arg-L-Thr-L-Tyr-)₄ -Lys₂ -Lys-Gly-OH,with different buffers and different amounts of sera.

For the solid phase synthesis of compounds of formula (I) it has beenused an automatic peptide synthesizer from Applied Biosystems (FosterCity, Calif., USA)--model 431A, software version 1.1--following thesynthesis procedure recommeded by the manufacturer and based onmethodology known and widely reported in the literature (Atherton andSheppard, 1988, Solid phase peptide synthesis: A practical approach, IRLPress, Oxford).

In the following examples "Cat. No." means catalogue number.

The following examples are given to better illustrate this inventionwithout limiting it in any way.

EXAMPLE 1

Solution preparation of a peptide of formula (I) (X₁ =Arg, X₂ =Tyr, n=1,R=OH) where the amino acids have D configuration

The synthesis started from the preparation of the protected dipeptideZ-D-Thr(tBu)-D-Tyr(tBu)-OMe to which subsequently was coupled, firstremoving the Z protecting group at the N-terminus, the protectedarginine amino acid to obtain the derivativeBoc-D-Arg(Pbf)-D-Thr(tBu)-D-Tyr(tBu)-OMe, which after completedeprotection led to peptide of formula (I).

a) Preparation of H-D-Tyr(tBu)-Ome (M.W. 251 amu)

To a suspension of H-D-Tyr(tBu)-OH (3.55 g, 15 mmoles, BachemFeinchemikalien, cat. No F-2170) in CH₃ OH (100 ml), chilled to -15° C.,there were added 3.54 g of SOCI₂ (30 mmoles, Aldrich, cat. No 23,046-4).After shaking 2 h at room temperature and 2 h at 90° C., the solvent wasremoved by evaporation and the residue dried over KOH for one night.There were thus obtained 4.21 g of crude product as hydrochloride salt(14.7 mmoles). Yield 98%.

b) Preparation of Z-D-Thr(tBu)-D-Tyr(tBu)-OMe (M.W. 541 amu)

To a solution of 7.21 g (10 mmoli) of Z-D-Thr(tBu)-OH (DCHA (BachemFeinchemikalien, cat. No C-1480) and 1.65 ml (16.2 mmoles) of N-methylmorpholine (NMM, Sigma, cat. No M-7889) in 75 ml of dry N-methylpyrrolidone (NMP), there were added drop-wise at -10° C., under shaking,2.12 ml (16.2 mmoles) of isobutylchloroformate (IBCF, Sigma, cat. No1-3253) diluted in 9 ml of CHCl₃. After 20 minutes, there was addeddrop-wise a solution of 4.21 g (14.7 mmoles) of H-D-Tyr(tBu)-OMe.sup.ΩHCl and 1.75 ml of NMM, in 75 ml of NMP. Then NMM (about 3 ml) wasfurther added to reach a pH of from 7.5 to 8.0.

The reaction mixture was kept under shaking for 2 h at 0° C. and thenovernight at room temperature.

The precipitated salts were filtered and the solution was concentratedby flash chromatography on silica gel using a mixture of AcOEt-hexane aseluent. The desired product (4.03 g; 7.45 mmoles), was obtained in theform of pure oil (TLC).

c) Preparation of H-D-Thr(tBu)-D-Tyr(tBu)-OMe (M.W. 407 amu)

4.03 g of Z-D-Thr(tBu)-D-Tyr(tBu)-OMe (7.45 mmoles) were dissolved in250 ml of CH30H. After addition of 850 mg of 10% Pd on activatedcharcoal (Fluka, cat. No 75990) a hydrogen stream was blown on thesolution at room temperature under shaking for 4 h. The hydrogenationreaction was monitored by TLC. After removal of the catalyst byfiltration and evaporation of the filtrate, 2.88 g (7.08 mmoles, yield95%) of H-D-Thr(tBu)-D-Tyr(tBu)-OMe were obtained in the form of pureoil (TLC).

d) Preparation of Boc-D-Arg(Pbf)-D-Thr(tBu)-D-Tyr(tBu)-OMe (M.W. 916amu)

A clear solution of 3.73 g (7.08 mmoles) of Boc-D-Arg(Pbf)-OH (BachemFeinchemikalien, cat. No A-3750) in 75 ml of dry NMP, comprising 0.85 ml(7.79 mmoles) of NMM was chilled to -10° C. under shaking. Then, 1.02 ml(7.79 mmoles) of IBCF diluted in 6 ml of CHCl₃ were added drop-wise.After 30 minutes, a solution of 2.88 g (7.08 mmoles) ofH-L-Thr(tBu)-L-Tyr(tBu)-OMe in 75 ml of CHCl₃ was added drop-wise in 20minutes. The reaction mixture was kept for 2 h at 0° C. and overnight atroom temperature. The solvent was evaporated under vacuum and the crudematerial was purified by flash chromatography on silica gel column,using a mixture of AcOEt-hexane as eluent. There were thus obtained 5.58g (6.08 mmoles, yield 86%) of Boc-D-Arg(Pbf)-D-Thr(tBu)-D-Tyr(tBu)-OMe,as pure oil (TLC and RP-HPLC).

e) Preparation of H-D-Arg-D-Thr-D-Tyr-OH (M.W. 438 amu)

The oil was treated for 2 h with 100 ml of a mixture having thefollowing composition

                  TABLE 1    ______________________________________    Component               % (v/v)    ______________________________________    Trifluoroacetic acid (TFA, Sigma Chem. Co.,                            83    St. Louis, Mo)    H.sub.2 O               4    Phenol (Sigma)          6    Thioanisol (Sigma)      5    Triisopropylsilane (TIS, Sigma)                            2    ______________________________________

The solution was concentrated to about 10 ml by vacuum evaporation ofthe trifluoroacetic acid, and the crude peptidic material wasprecipitated by addition of 150 ml of cold ethyl ether. After removal ofthe precipitating agent, a subsequent washing with 100 ml of cold ethylether was carried out to better solubilize the scavengers. All thepeptidic material, was dissolved in 50 ml of H₂ O/CH₃ CN/TFA 50/50/0.1and then frozen and lyophilized.

1.89 g of the tripeptide H-D-Arg-D-Thr-D-Tyr-OH, equivalent to 4.32mmoles, were thus recovered. Yield, 71%. RP-HPLC analysis of a 5 μgaliquot the product showed that it was 97% pure.

Working in a similar way the following additional compounds have beenprepared:

H-L-Arg-L-Thr-L-Tyr-OH

H-L-Tyr-L-Thr-L-Arg-OH

H-D-Tyr-D-Thr-D-Arg-OH

Preparation yields, final purity and experimentally observed molecularweigth as determined by mass spectrometry are shown in the followingTable A.

                                      TABLE A    __________________________________________________________________________                               Observed                                    Theoretical    Compound         Yield (%)                          Purity (%)                               M.W. M.W.    __________________________________________________________________________    H--D--Arg--D--Thr--D--Tyr--OH                     71   97   438.3                                    438    H--L--Arg--L--Thr--L--Tyr--OH                     79   97   438.7                                    438    H--L--Tyr--L--Thr--L--Arg--OH                     76   96   438.1                                    438    H--D--Tyr--D--Thr--D--Arg--OH                     77   97   438.5                                    438    __________________________________________________________________________

EXAMPLE 2

Solid phase preparation of a peptide of formula (I) (X₁ =Arg, X₂ =Tyr,n=4, R=Lys-Lys(εLys)-Gly) where the amino acids have L configuration.

The peptide preparation was carried out in the solid phase using anautomatic peptide synthesizer from APPLIED BIOSYSTEMS Mod 431 Afollowing the Fmoc/HOBt/DCC methodology (Konig, W., and Geiger, R.,1970, Chem. Ber. 103, 788-798.) and following protocols as recommeded bythe manufacturer (APPLIED BIOSYSTEM, USA).

The preparation was carried out a 0.1 mmole scale starting from aacid-labile resin for peptide synthesis prederivatized with glycine(chlorotritylichloridric NOVOBIOCHEM Cat No. 04-12-2800, 0.1 mmole)protected at the N-terminal amino group with the Fmoc group, which, inthe first synthesis cycle, was deprotected by treatment with 3.0 ml ofpiperidine (20 % in N-methyl-2-pyrrolidone (ABI Cat No. 400629) for 14minutes, at room temperature under stirring.

The deprotected resin was then washed 5 times with 2.5 ml ofN-methyl-2-pyrrolidone (Merck Cat. No. 806072) for 9 minutes undershaking at room temperature.

Then the amino acid Fmoc-Lys(Fmoc) (Novabiochem Cat. No. 04-12-1085)acid was attached, previously transformed into the correspondingbenzotriazole compound active ester by incubation with a solution ofhydroxybenzotriazole (HOBt, APPLIED BIOSYSTEMS, Cat. No 400662) anddicyclohexylcarbodiimide (DCC, APPLIED BIOSYSTEMS, Cat. No 400663).After removal of the Fmoc protecting groups in α and ε position, asecond coupling of Fmoc-Lys(Fmoc) (NOVABIOCHEM Cat. No. 04-12-1085) wasaccomplished, to form the central tetrameric core R. ThenFmoc-Tyr(tBu)-OH (BACHEM FEINCHEMIKALIEN Cat. No B-1255) acid wasattached, previously transformed into the corresponding benzotriazolecompound active ester by incubation with a solution ofhydroxybenzotriazole (HOBt, APPLIED BIOSYSTEMS, Cat No 400662) anddicyclohexylcarbodiimide (DCC, APPLIED BIOSYSTEMS, Cat No 400663).

The suspension resin/activated amino acid was shaken for 51 minutes.During the coupling of the tyrosine residue the activation of thethreonine residue was accomplished. 1 mmole of Fmoc-Thr(tBu)-OH (BACHEMFEINCHEMIKALIEN Cat No B-1245) was transformed in the correspondingbenzotriazole compound active ester by incubation with a solution ofhydroxybenzotriazole (HOBt, APPLIED BIOSYSTEMS, Cat No 400662) anddicyclohexylcarbodiimide (DCC, APPLIED BIOSYSTEMS, Cat No 400663).

When the coupling of tyrosine was over, the resin was extensively washedwith N-methyl-pirrolidone (NMP). Removal of Fmoc group was accomplishedby treatment with 3 ml of 20% piperidine in NMP. After several washeswith NMP, the amino acid threonine previously activated, was transferredon the resin. The coupling reaction lasted for 51 minutes, and duringthat time the third amino acid arginine was activated. The derivativeFmoc-Arg(Pbf)-OH (BACHEM FEINCHEMIKALIEN Cat No B-2375) was used. Theactivation method was the same as described in relation to threonineactivation.

After threonine acylation and subsequent removal of the Fmoc group withpiperidine, activated arginine was transferred on the resin for thecoupling step. After the required washings for removing the amino acidexcess and the deprotection of the Fmoc groups even on the arginineresidue, the resin was washed extensively first with NMP and then withcichloromethane (DCM). At the end, the resin was dried by an argonstream. 215.8 mg of resin were recovered.

A fully protected tetramer of formula

    (H.sub.2 N-Arg(Pmc)-Thr(OtBu)-Tyr(OtBu)-CO).sub.4 -Lys.sub.2 -Lys-Gly-OH

has been prepared by treating the resin with a mixture of acetic acid(Merck Cat. No. 63), dichloro methane (Merck Cat. No. 6050), and ethanol(Merck Cat. No. 8006) in the ratio 80:10:10 v/v.

This treatment allows the detachment of the peptide from the resin butnot of the amino acid side chain protecting groups.

Alternatively, a fully deprotected tetramer of formula

    (H.sub.2 N-Arg-Thr-Tyr-CO).sub.4 -Lys.sub.2 -Lys-Gly-OH

has been obtained by treating the resin with 10 ml of a mixture oftrifluoroacetic acid and scavengers having the composition shown inTable 1, Example 1.

The solution, was concentrated to about 1 ml by evaporation of thetrifluoroacetic acid under vacuum. The crude peptidic material wasprecipitated by adding 30 ml of cold ethyl ether. After removal of theprecipitation agent, a second washing with 30 ml of cold ethyl ether wascarried out to further solubilize the scavengers. All the peptidicmaterial, dissolved in 5 ml of H₂ O/CH₃ CN/TFA 50/50/0.1, was frozen andlyophilized.

103.8 mg of the tetrameric tripeptide (H-L-Arg-L-Thr-L-Tyr-)₄ -Lys₂-Lys-COOH were recovered. RP-HPLC analysis of a 5 μg aliquot of theproduct showed a purity close to 97%.

Working in a similar way there were prepared the additional compoundsshown in the Table B.

                                      TABLE B    __________________________________________________________________________                                         Observed                                              Theoretical    Compound                   Yield (%)                                    Purity (%)                                         M.W. M.W.    __________________________________________________________________________    (H--D--Arg--D--Thr--D--Tyr--).sub.4 --Lys.sub.2 --Lys--GlyOH                               75   97   438.3                                              2142    (H--L--Arg--L--Thr--L--Tyr--).sub.4 --Lys.sub.2 --Lys--Gly--OH                               82   97   438.7                                              2142    (H--L--Tyr--L--Thr--L--Arg--).sub.4 --Lys.sub.2 --Lys--Gly--OH                               79   96   438.1                                              2142    (H--D--Tyr--D--Thr--D--Arg--).sub.4 --Lys.sub.2 --Lys--Gly--OH                               81   97   438.5                                              2142    __________________________________________________________________________

EXAMPLE 3

Immobilization of peptides of Examples 1 and 2 on Activated CH-Sepharose4B and purification of immunoglobulins from sera by affinitychromatography.

The peptide of formula (H-L-Arg-L-Thr-L-Tyr-)₄ -Lys₂ -Lys-Gly-OH (5 mg)was dissolved in 5 ml of 0.1M sodium bicarbonate buffer pH 9.0 and thenadded to 1.2 g of activated resin CH-Sepharose 4B (Pharmacia, Uppsala,Sweden Cat. No. 17-0490-01), which is a chromatographic support foraffinity chromatography preactivated for the direct coupling to peptidesand proteins. The suspension was shaken for 24 h and the coupling levelwas monitored by taking aliquots of the reaction mixture at differenttimes and subsequent RP-HPLC analysis.

Approximately 90 % of initial peptide resulted covalently linked to theresin after 24 h. The derivatized resin was washed with 50 ml of 1M TRISpH 9.0 and then packed on a glass column (100×6.6 mm I.D.).

Working in a similar way with other compounds of this invention, therewere obtained the immobilization yields shown in Table C.

                  TABLE C    ______________________________________                                 Im-                                 mobili-                                 zation                                 Yield    Compound                     (%)    ______________________________________    (H--D--Arg--D--Thr--D--Tyr--).sub.4 --Lys.sub.2 --Lys--GlyOH                                 89    (H--L--Arg--L--Thr--L--Tyr--).sub.4 --Lys.sub.2 --Lys--Gly--OH                                 90    (H--L--Tyr--L--Thr--L--Arg--).sub.4 --Lys.sub.2 --Lys--Gly--OH                                 85    (H--D--Tyr--D--Thr--D--Arg--).sub.4 --Lys.sub.2 --Lys--Gly--OH                                 89    H--D--Arg--D--Thr--D--Tyr--OH                                 95    H--L--Arg--L--Thr--L--Tyr--OH                                 93    H--L--Tyr--L--Thr--L--Arg--OH                                 96    H--D--Tyr--D--Thr--D--Arg--OH                                 94    ______________________________________

In order to purify immunoglobulins, the column was equilibrated with a25 mM BIS-TRIS buffer (SIGMA, Cat. B9754) pH 6.5, at a flow rate of 1ml/min, while the eluent was monitored at 280 nm. One milliliter ofcrude rabbit serum (SIGMA Cat. No. R 9133) was then loaded on thecolumn, and after elution of non retained material at the column voidvolume, the eluent was changed to 0.1M acetic acid.

Material desorbed by such a treatment was collected and analyzed byelectrophoretic analysis on a polyacrylamide gel.

The purification of rabbit immunoglobulins from crude serum by affinitychromatography is shown in FIG. 1 while the electrophoretic analysis ofthe collected fractions is shown in FIG. 2. As clearly shown by theelectrophoretic analysis, the column was able to retain theimmunoglobulin fraction from the crude serum, while albumin was notretained and was eluted at the column void volume. Furthermore, in FIG.2 are also shown the electrophoretic analyses corresponding to severalrabbit immunoglobulins purifications obtained after equilibration of theaffinity column with different buffers, namely 0.1M ammonium acetate pH5.7 (A), 0.1M sodium phosphate pH 7.0 (B), or 0.1M sodium phosphate pH8.5 (C). As clearly shown by the electrophoretic analysis of thefractions desorbed by treatment with acetic acid 0.1M, in all the threecases an optimal purification of immunoglobulins from contaminants wasachieved.

Furthermore, the column showed a remarkable purification capability,allowing purification of immunoglobulins from 0.5 ml (E), 1 ml (F), and1.5 ml (G) of serum. The column selectivity resulted to be superior tothat of columns with immobilized protein A. Indeed, purification of thesame serum on columns of immobilized protein A having the samedimensions provided purified fractions always comprising traces ofalbumin. The purification capability of both monomeric and multimericpeptides under examination and analogs thereof prepared with aminoacidsin configuration L or D resulted to be similar and did not depend on thetype of affinity chromatography support which was used. Indeed similarresults were attained with other supports such as Protein-Pak (Waters,USA), Eupergit C30N (Sigma, USA) and Affi-Gel (Bio-Rad, USA).

Similar results were obtained with columns prepared with immobilizedpeptides of formula (1) in the isolation of IgG from mouse, rat, goat,sheep, horse, human, and bovine sera, as well as of chicken IgY, humanIgA, and human IgM, from crude sources.

    __________________________________________________________________________    SEQUENCE LISTING    <160> 4    <210> 1    <211> 4    <212> PRT    <213> Artificial Sequence    <220>    <223> Description of Artificial Sequence:Synthetic    <400> 1    GlyGlyGlyGly    <210> 2    <211> 5    <212> PRT    <213> Artificial Sequence    <220>    <223> Description of Artificial Sequence:Synthetic    <400> 2    GlyGlyGlyGlyGly    15    <210> 3    <211> 5    <212> PRT    <213> Artificial Sequence    <220>    <223> Description of Artificial Sequence:Synthetic    <400> 3    GlyAlaGlyAlaGly    15    <210> 4    <211> 7    <212> PRT    <213> Artificial Sequence    <220>    <223> Description of Artificial Sequence:Synthetic    <400> 4    AlaGlyAlaGlyAlaGlyAla    15    __________________________________________________________________________

We claim:
 1. A compound of formula

    (H.sub.2 N-X.sub.1 -Thr-X.sub.2 -CO).sub.4 -Lys.sub.2 -Lys-Gly-OH (1A)

where X₁ and X₂, different from one another, are amino acid residuearginine or tyrosine in configuration L or D, wherein the hydroxy groupof threonine and tyrosine and the guanidine moiety of arginine areoptionally protected by a protecting group conventionally used inpeptide chemistry for protecting the hydroxy group and the guanidinemoiety, respectively.